Neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body disease (LBD), amyotrophic lateral sclerosis (ALS) and prion diseases (PrD), while pathologically different, can undergo similar abnormal metallochemical reactions between proteins and the redox-active metal ions copper or iron. These reactions damage cellular components and result in abnormal protein aggregation through the generation of reactive oxygen species (ROS) which include free radicals such as superoxide anion and hydroxyl radical and other molecular species such as hydrogen peroxide and peroxynitrite (Droge and Schipper (2007) Aging Cell, 6:361-70; Bush and Goldstein (2001) Novartis Found. Symp., 235:26-43; Moreira et al. (2005) J. Neural. Transm., 112:921-32; Butterfield and Kanski (2001) Mech. Ageing Dev., 122:945-62; Wisniewski and Sigurdsson (2007) Febs J., 274:3784-98).
Cellular ROS are generated as a side product in mitochondria because of incomplete metabolic reduction of molecular oxygen (O2) to water. Common ROS include the superoxide anion (O2−), the hydroxyl radical (OH.), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Superoxide anions are continuously formed in the mitochondria as molecular oxygen (O2) acquires an additional electron. Hydroxyl radicals are the most reactive and damaging generated ROS. They are predominantly formed by a Fenton reaction between transitions metals (usually iron(II) or copper(I)) and hydrogen peroxide; however, it can also be formed through the Haber-Weiss reaction of superoxide anion and hydrogen peroxide. Although these metals are oxidized in this process, they are returned to their “active” (reduced) state through a process of ‘redox cycling’ with vitamin C or other cellular reductants. Hydrogen peroxide, produced in vivo through several reactions, can either be converted to the highly reactive and damaging hydroxyl radicals or converted to water. It is formed by the reduction of superoxide radical by superoxide dismutase and reduced to water by either catalase or glutathione peroxidase. Hydrogen peroxide can also form singlet oxygen. While not a free radical, singlet oxygen is highly reactive because it can serve as a catalyst for free radical formation by transferring its energy to other molecules.
ROS damages cellular components by oxidizing proteins, lipid bilayers, and DNA. This can result in alterations of protein conformations and enzyme activities. With polyunsaturated fatty acids, ROS can generate lipid peroxides which subsequently can oxidize adjacent unsaturated fatty acids in a chain reaction event that leads to the disruption of plasma membranes and membranes of cellular organelle components, such as the mitochondria. Characteristic break-down products of lipid oxidation include 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). Oxidation of DNA results in strand breaks, DNA-protein cross-linking, and base modifications that can lead to mutations impacting DNA replication. Cells possess a variety of enzymatic and non-enzymatic antioxidant systems to protect against ROS damage. However, several of these protective enzymes, such as cytosolic copper-zinc superoxide dismutase (CuZnSOD) and mitochondrial manganese superoxide dismutase (MnSOD), contain metals. As a result, cells must maintain a delicate balance between free and bound pro-oxidant versus antioxidant metal ions that are critical to cellular homeostasis. The aging brain has a progressive imbalance between antioxidant defenses and intracellular concentrations of ROS.
Oxidative stress results from an imbalance between biochemical processes leading to the production of ROS and those responsible for the removal of ROS, the so-called antioxidant cascade. Oxidative stress increases with age and prolonged tissue exposure to oxidative stress results in cellular damage that eventually leads to cell death. Neural tissues of the brain are especially susceptible to ROS and oxygen free radical activity has been observed in the brain hippocampus, substantia nigra, caudate putamen and in the spinal fluid. The increased susceptibility of the neural tissues is due to their higher metabolic rates, high compositions of peroxidation susceptible fatty acids, high intracellular concentrations of transition metals capable of catalyzing Fenton reactions, low levels of antioxidants, and a reduced capability of tissue regeneration. In addition to the mitochondrial generation of ROS, neural tissues possess brain-specific oxidases such as monoamine oxidase that also generate hydrogen peroxide. ROS also results from neuroinflammatory responses induced by reactive microglia, macrophages and proinflammatory T-cells.
Many consider oxidative damage to be a hallmark of neurodegenerative disorders and the relationship between ROS and neurodegenerative disorders has been extensively reviewed (Floyd, R. A. (1999) Proc. Soc. Exper. Biol. Med., 222:236-45; Doraiswamy and Finefrock (2004) Lancet Neurology, 3:431-4; Reynolds et al. (2007) Int. Rev. Neurobiol., 82:297-325; Andersen, J. K. (2004) Nature Med., 10:S18-25; Sayre et al. (2005) Ann. Ist. Super Sanita, 41:143-64; Casadesus et al. (2004) J. Alzheimers Dis., 6:165-9; Gaggelli et al. (2006) Chem. Rev., 106:1995-2044). With normal ageing, the brain accumulates metals ions such iron (Fe), copper (Cu), and zinc (Zn) and a major focus of studies on the generation of neurodegenerative ROS has been the involvement of the redox-reactive metals.
Metal-protein associations with Cu(II), Fe(III), or Mn(II) can also lead to protein aggregation. Cu, Zn and Fe accumulate in β-amyloid (Aβ) deposits in the brains of patients with AD (Lovell et al. (1998) J. Neurol. Sci., 158:47-52). Both the amyloid precursor protein and Aβ bind and reduce Cu. Cu binding to Aβ promotes the aggregation of Aβ into metal-enriched precipitates (plaques) and the abnormal combination of Aβ with Cu or Fe induces the production of hydrogen peroxide (Smith et al. (2007) Biochim. Biophys. Acta, 1768:1976-90; Bush, A. I. (2002) Neurobiol. Aging, 23:1031-8). Addition of Zn to synthetic Aβ induces protease resistant aggregation and precipitation of the synthetic Aβ (Barnham et al. (2006) Trends Biochem. Sci., 31:465-72; Huang et al. (1997) J. Biol. Chem., 272:26464-70). Cu can also bind to α-synuclein, a protein observed to aggregate in Lewy Bodies of PD. While its present role in neurodegeneration is undefined and controversial, copper chelators appear to prevent its aggregation (Brown, D. R. (2007) FEBS J., 274:3766-74). Regardless, metal chelation has been proposed for the treatment of neurodegenerative disorders such as Parkinson's Disease and Alzheimer's (Gaeta et al. (2006) Br. J. Pharmacol., 146:1041-1059; Kaur et al. (2002) Aging Cell 1:17-21; Gouras et al. (2001) 30:641-642).
While the relationship(s) between neurodegeneration, ROS and redox-reactive metal ions have not been clearly defined, studies suggest that targeting oxidative pathways may be therapeutic (Smith et al. (2007) Biochim. Biophys. Acta, 1768:1976-90). A wide variety of antioxidants have been examined to reduce ROS. These range from natural products with antioxidant properties such as aged garlic extract, curcumin, melatonin, resveratrol, Ginkgo biloba extract, green tea, vitamin C, L-caritine, vitamin E, and cannabinoids to derivatives of lipoic acid, analogs of Coenzyme Q (MitoQ), and the “thiol-delivering” glutathione-mimics such as tricyclodecan-9-yl-xanthogenate (Frank and Gupta (2005) Ann. Clin. Psych., 17:269-86; Garcia-Arencibia et al. (2007) Brain Res., 1134:162-70; Bolognesi et al. (2006) Mini Rev. Med. Chem., 6:1269-74; Binienda et al. (2005) Annal. NY Acad. Sci., 1053:174-82; Tauskela et al. (2007) Idrugs, 10:399-412; Perluigi et al. (2006) Neuroscience, 138:1161-70).
Chelation of redox-active metals is another promising approach to reduce the generation of ROS. A number of structurally diverse chelators have been evaluated (desferrioxamine (DFO), clioquinol, JKL 169, D-penicillamine, DP-109, VK-28, epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG), epicatechin (EC), epigallocatechin (ECG), H2GL1, H2GL2, M-30); however, the hydrophobic natures of many of these chelators hinder their ability cross the BBB. Desferrioxamine, an iron specific chelator with high affinity for Cu, Zn and Al, has been reported to decrease the progression of Alzheimer's disease (McLachlan et al. (1991) Lancet, 337:1304-8; McLachlan et al. (1993) Ther. Drug Monit., 15:602-7). However, this compound is not orally active and does not significantly cross the BBB (Cuajungco and Lees (1998) Brain Res., 799:97-107; Finefrock et al. (2003) J. Am. Geriatr. Soc., 51:1143-8). Clioquinol is an orally active antibiotic that also chelates metals. Through chelation, it reduces Cu uptake and counteracts Cu efflux activities of the amyloid precursor protein of AD (Treiber et al. (2004) J. Biol. Chem., 279:51958-64), disaggregates the metal ion-induced aggregates of Aβ(1-40), and retards fibril growth through Zn(II)-clioquinol complex formation (Raman et al. (2005) J. Biol. Chem., 280:16157-62). Its efficacy has been demonstrated in vitro, in vivo in animal models, and in several small clinical trials where statistically significant results were seen in the more severely affected subgroups of AD patients (Jenagaratnam and McShane (2006) Cochrane Database Of Systematic Reviews; Smith et al. (2007) Biochim. Biophys. Acta, 1768:1976-90; Crouch et al. (2006) Drug News & Perspect., 19:469-74; Rose and Gawel (1984) Acta Neuro. Scand. Supp., 100:137-45). JKL169 is a 14-membered saturated tetramine that when injected into rats has been reported to demonstrate activity similar to clioquinol in reducing Cu levels in brain cortex and maintaining normal Cu levels in the blood, CSF and corpus callossum (Moret et al. (2006) Bioorg. Med. Chem. Lett., 16:3298-301). DP109 is a lipophilic chelator that reduced the levels of aggregated insoluble Aβ and conversely increased soluble forms in transgenic mice (Lee et al. (2004) Neurobiol. Aging, 25:1315-21). D-penicillamine, which also chelates copper, has been reported to delay the onset of prion disease in mice infected with scrapie (Sigurdsson et al. (2003) J. Biol. Chem., 278:46199-202).
Recent reports have also focused on the potential treatment of neurodegenerative diseases by combining chelation with antioxidant/neuroprotective therapy. For example, green tea extract (catechins) and its major component, EGCG, possess divalent metal chelating, antioxidant, and anti-inflammatory activities (Mandel et al. (2006) Mol. Nutr. Food Res., 50:229-34). In addition, catechins may modulate signal transduction pathways, cell survival/death genes and mitochondrial function (Mandel et al. (2005) Neurosignals, 14:46-60). Both have been reported to prevent striatal dopamine depletion in mice as well as substantia nigra dopaminergic neuron loss induced by the PD-inducing neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Levites et al. (2001) J. Neurochem., 78:1073-82). Similarly neuroprotective affects have been observed with in vitro studies with extracts from anthocyanin rich Vaccinium species (Yao and Vieira (2007) Neurotoxicology, 28:93-100). Clinical efficacy of neither green tea constituents nor other flavonoids has been established. Several novel antioxidant-iron chelators bearing 8-hydroxyquinoline moiety (VK-28 and M30) have also been synthesized and evaluated for their ability to chelate iron and possess neuroprotective activity (Zheng et al. (2005) Bioorg. Med. Chem., 13:773-83). In vitro, these compounds were able to chelate Fe, scavenge hydroxyl radicals, and inhibit monoamine oxidase (MAO) (Zheng et al. (2005) J. Neurochem., 95:68-78). In vivo, both inhibited MAO with M30 demonstrating brain selective (striatum, hippocampus and cerebellum) irreversible MAO-A and -B inhibition in mice (Gal et al. (2005) J. Neurochem., 95:79-88). The synthesis of two multifunctional carbohydrate-containing compounds N,N′-bis[(5-β-D-glucopyranosyloxy-2-hydroxy)-benzyl]-N,N′-dimethylethane-1,2-diamine (H2GL1) and its t-butyl-analog (H2GL2) have recently been reported (Storr et al. (2007) J. Am. Chem. Soc., 129:7453-7463). Initial in vitro studies suggest that both of these water soluble, carbohydrate-containing compounds have significant antioxidant capacity and moderate affinity for Cu(II) and Zn(II). While in vivo studies have not been reported, sugar moieties have been added to these molecules in anticipation that these water soluble compounds will utilize GLUT transporters to cross the BBB.
ROS and the Fenton reaction as well as the presence of metals such as Fe and Cu have also been implicated in cataracts and macular degeneration. Indeed, it has been well established that cataract and macular degeneration are initiated by oxidation and oxidative stress. With regard to cataracts, it has also been shown that Fe and Cu levels in serum are increased in patients with psuedoexofoliative cataracts (Cumurcu et al. (2006) 16:548-52) and cataracts has also been associated with hyperferritinemia (Ismail et al. (2006) 16:153-160; Vanita et al. (2006) Mol. Vis., 12:93-99; Roetto et al. (2002) 29:532-535). It has also been determined that lens levels of Cu in diabetic patients is significantly higher compared to non-diabetic patients and that tobacco smoke increases lens Fe levels (Dawczynski et al. (2002) Biol. Trace Elem. Res., 15-24; Avunduk et al. (1997) Exp. Eye Res., 65:417-423). With regard to macular degeneration, iron induced oxidative damage has been identified as a potential factor in macular degeneration as Fe and Zn levels and the iron carrier transferrin have been shown to be increased in eyes with macular degeneration (Chowers et al. (2006) Invest. Opthalmol. Vis. Sci., 47:2135-2140; Dunaief, J. L. (2006) Invest. Opthalmol. Vis. Sci., 47:4660-4664; Lengyel et al. (2007) 84:772-780). Macular degeneration has also been observed in a patient with aceruloplasminemia, a disease associated with retinal iron overload (Dunaief et al. (2005) Opthalmol., 112:1062-1065). Additionally, it has been noted that maculas affected by macular degeneration contain increased chelatable iron in the retinal pigment epithelium and Bruch's membrane (Hahn et al. (2003) 121:1099-1105).